Chapter 9 DNA and Its Role in Heredity

Scientists found that DNA:

(1) is present in the cell nucleus and in condensed chromosomes, (2) doubles in abundance in the cell during S phase of the cell cycle, and (3) is twice as abundant in the diploid cells as in the haploid cells of a given organism.

Initiation of DNA Replication in Prokaryotes and Eukaryotes

(A) Initiation of DNA replication in most prokaryotes occurs at the single ori site. (B) Initiation in eukaryotes involves many—up to tens of thousands—of ori sites on each chromosome. (C) Initiation of replication in both prokaryotes and eukaryotes involves several proteins.

Watson and Crick Described the Structure of DNA

(A) James Watson (left) and Francis Crick and their DNA model in 1953. (B) A space-filling model showing the atoms in DNA. (nm, nanometers.) (C)The detailed chemical structure of DNA. Only N atoms involved in H-bonding are shown.

Rosalind Franklin and Her X-Ray Diffraction Image of DNA

(A) The positions of atoms in a crystallized chemical substance can be inferred by the pattern of diffraction of X rays passed through it. The pattern in DNA is both highly regular and repetitive. (B) Rosalind Franklin's famous "photograph 51," shown here, helped other scientists determine the structure of the DNA molecule.

The errors by DNA polymerase do indeed occur. But most of them are repaired, either as they happen or shortly afterward. There are two ways in which this occurs:

1) Proofreading is performed by the DNA polymerases during replication. 2) Mismatch repair occurs after DNA has been replicated.

As we will see, DNA replication involves several different proteins. The overall process occurs in three steps:

1. Initiation 2. Elongation 3. Termination

The double-helical structure of DNA suggested a way in which the information in DNA might be copied (replicated) so that it could be passed down to cells produced in mitosis and meiosis.

Because of complementary base pairing, the information contained in a DNA molecule is fully contained in each of the two strands. Thus knowing the identity of a particular base on one strand (e.g., adenine), means that the base on the complementary strand is also known (e.g., thymine).

The chemical composition of DNA:

Biochemists knew that DNA is a very long polymer of four different nucleotides. Each of these nucleotides consists of a molecule of the sugar deoxyribose, a phosphate group, and a nitrogen-containing base.

induced mutations

Changes in the sequence of DNA caused by a mutagen. (Compare with spontaneous mutation.)

mutagens

Most cancer-causing mutations are caused by mutagens, substances that chemically alter DNA and lead to induced mutations. -Any agent (e.g., a chemical, radiation) that increases the mutation rate. -Cigarette smoke contains mutagens that chemically alter the bases in DNA and change their base-pairing properties, leading to mutations. -Mutagens often cause DNA damage, which is an alteration of the molecule so that it has a chemical structure not normally found in DNA. -Both of these types of (sunlight and radiation of power plants) radiation damage DNA and can cause mutations.

leading strand

One of these template strands—the leading strand—is synthesized continuously, while the other—the lagging strand—is synthesized discontinuously. The leading template strand is oriented so that DNA polymerase can add nucleotides to the 3′ end of the new strand in the same direction as fork movement and so polymerization occurs continuously.

Primer replacement

Primer replacement involves a different DNA polymerase than is used for elongation.

Termination

Termination, in which synthesis ends after each region of the DNA molecule has been replicated

DNA ligase

This single-strand break, or nick, is fixed by the enzyme DNA ligase, which catalyzes the formation of a phosphodiester bond, linking the fragments and making the lagging strand whole.

topoisomerase

Topoisomerase works by breaking the sugar-phosphate backbone of one strand, allowing the strand to untwist and then rejoining the backbone.

DNA replicates semiconservatively

Watson and Crick suspected each strand of a DNA double helix could be used as a template to synthesize a new strand of DNA using complementary base pairing.

9.2 DNA Replication Is Semiconservative

It is crucial that DNAis replicated both completely and accurately during the cell cycle. As mentioned above, Watson and Crick noted that the double-helical model of DNA suggested an obvious means of replication.

Mismatch repair

Mismatch repair occurs after DNA has been replicated. The mismatch repair complex scans the newly replicated molecule looking for mismatched base pairs. It recognizes these because they have abnormal hydrogen bonding and, in many cases, alter the width of the DNA helix (when the mismatch involves two purines or two pyrimidines). If a mismatch is found, the mismatch repair proteins make two single-strand cuts on the new strand and remove the intervening fragment, and then a DNA polymerase resynthesizes the missing piece and DNA ligase seals the remaining single-strand break (FIGURE 9.15B). The mismatch repair complex finds and correctly repairs 99 percent of mismatches that escape DNA polymerase proofreading. You can think of this process as akin to a good editor or professor at the writing center.

Chemical changes in bases can lead to DNA mutations

Replication errors by DNA polymerases are just one way in which mutations can occur in DNA. -Other mechanisms involve spontaneous or induced chemical changes in the DNA molecule. Bases in DNA constantly undergo spontaneous chemical changes at a low rate that can alter base-pairing properties and result in point mutations.

Review

-As we noted in Key Concept 3.3, a free nucleotide can have one, two, or three phosphate groups attached to its pentose sugar. -The nucleotides used for DNA synthesis each have three phosphates attached at the 5′ carbon of the deoxyribose, and are thus called deoxyribonucleoside triphosphates, or dNTPs, where N indicates the specific nucleoside and is either adenosine (A), thymidine (T), cytidine (C) or guanosine (G).

Eukaryotic vs. Prokaryotic

-Eukaryotic chromosomes are much longer than those of prokaryotes—up to a billion base pairs—and are linear, not circular. -In addition, their replication forks move much more slowly, on the order of tens of bases per second. -So eukaryotic chromosomes have multiple oris, scattered at intervals of 10,000-40,000 base pairs, allowing them to replicate their chromosomes in a few hours or less

Crucial evidence for the three-dimensional structure of DNA was obtained using X-ray crystallography

-Some chemical substances, when they are isolated and purified, can be made to form crystals. -The positions of atoms in a crystallized substance can be inferred from the diffraction pattern of X rays passing through the substance.

Important

-Thus the surfaces of the base pairs are chemically distinct from one another both in identity and spatial orientation, allowing other molecules, especially proteins, to recognize specific base-pair sequences and bind to them. -The atoms and groups in the major groove are more accessible and tend to bind other molecules more frequently than those in the minor groove. -This binding of proteins to specific base-pair sequences is the key to protein-DNA interactions, which are necessary for the replication and expression of the genetic information in DNA

Some spontaneous changes:

-When a base temporarily forms its rare tautomer (a tautomeric shift) (FIGURE 9.17A) it can pair with a different base, leading to mismatches during DNA replication. The resulting mismatched base pair (e.g., A−C instead of A−T) produces a mutation after a second round of replication. -Another spontaneous change is the occasional removal of an amino group (NH2) in cytosine, producing uracil (FIGURE 9.17B). If this deamination process occurs during, or just prior to DNAreplication, an A will be inserted into the new DNA strand (because A pairs with U) instead of G (which pairs with the original C). Again, mutation will result after a second round of replication.

"end-replication problem"

-there is an RNA primer on the 5′ end of the new strand. While there are enzymes that can remove the RNA primer, no DNA can be synthesized to replace the resulting gap because there is no 3′ end to extend. -So the new chromosome has a short region of single-stranded DNA overhang at one end. -This situation activates a mechanism for cutting off the single-stranded region, along with some of the intact double-stranded DNA. -Thus the chromosome becomes slightly shorter with each cell division. -This shortening is termed the "end-replication problem."

To understand the difference between leading and lagging strand synthesis, it is important to remember two things:

1) The two DNA strands are antiparallel—that is, the 3′ end of one strand is paired with the 5′ end of the other. 2) The DNA polymerase synthesizes new DNA by adding nucleotides only to the 3′ end of a new strand.

Such staining experiments supported two other predictions for DNA as the genetic material:

1) Virtually all nondividing somatic cells of a particular organism have the same amount of nuclear DNA. This amount varies from species to species. 2) Cells resulting from meiosis have half the amount of nuclear DNA as somatic cells.

Biologists proposed three different models of replication based on how the original and newly synthesized strands would be arranged following replication of a DNA molecule:

1. Semiconservative replication 2.Conservative replication 3. Dispersive replication

Mismatches Can Lead to Base-Pair Substitutions in DNA

A base-pair substitution is a mutation that exchanges one base pair for another. (A) In some cases, mismatch repair removes the old strand, instead of the new, resulting in a base-pair substitution. (B) In other cases, the mismatch may not be repaired before the next round of replication, again resulting in a base-pair substitution.

diffraction pattern

A diffraction pattern can be visualized on a photographic plate as closely spaced light and dark spots or bands, formed when X rays are bent by objects (such as atoms) in their path. A diffraction pattern obtained by Rosalind Franklin in the early 1950s provided enormous insight into the structure of DNA.

Spontaneous mutations

A genetic change caused by internal cellular mechanisms, such as an error in DNA replication. (Contrast with induced mutation.) -Spontaneous mutations caused by polymerase errors or spontaneous chemical changes in bases cannot be avoided, though their rate is quite low.

template

A molecule or surface on which another molecule is synthesized in complementary fashion, as in the replication of DNA.

Nucleotides Are Added to the 3′ End during DNA Synthesis

A nucleotide that complements the template base is added to a growing DNA strand at the 3′ end. The new strand thus extends in the 5′-to-3′ direction. Energy for the formation of the phosphodiester bond between nucleotides comes from the exergonic cleavage of the pyrophosphate from the triphosphate of the incoming nucleotide. The template strand is read in the 3′-to-5′ direction.

polymerase

A polymerase is an enzyme that synthesizes nucleic acid polymers. RNA polymerase and DNA polymerase are used to assemble RNA and DNA molecules, respectively. In the case of primase, it is an RNA polymerase that synthesizes RNA, using a DNA template.

More Info

After 20-30 cell divisions, the chromosome ends become short enough that telomeres can no longer fulfill their protective role. Apoptosis (programmed cell death) ensues, and the cell dies.

bacteriophages

Any of a group of viruses that infect bacteria. Also called phage. -Many viruses, including bacteriophages (viruses that infect bacteria), are composed of DNA and only one or a few kinds of protein. -Experiments performed by Alfred Hershey and Martha Chase showed that when a bacteriophage infects a bacterial cell, it injects only viral DNA. The injected DNA is then used to make progeny bacteriophage, implying that the information to do so is encoded on DNA.

telomerase

At every cell division, the number of telomere repeats is reduced at one chromosome end or the other. However, there is an enzyme called telomerase that can add DNA repeats. -Telomerase is yet another DNA polymerase: in this case, it uses an RNA template to synthesize new DNA. The RNA template is a component of the telomerase enzyme.

The Life Cycle of Bacteriophage

Bacteriophage T2 infects Escherichia coli and depends on the bacterium to produce new viruses. The bacteriophage consists of DNA contained within a protein coat. When the virus infects an E. coli cell, it injects its genetic material into the host bacterium, turning the host cell into a viral replication machine.

Experimental evidence confirmed that DNA is the genetic material

Chromosomes in eukaryotic cells contain DNA, but they also contain proteins that are bound to DNA. Therefore it was difficult to rule out the possibility that genetic information might be carried in proteins.

9.3 DNA Mutations Alter DNA Sequence

DNA replication is not perfect, however: errors do occur during the process and may result in mutations—permanent, inherited changes in DNA sequence. DNA is also subject to damage by chemicals and other environmental agents, which can also lead to mutations.

origins of replication (ori)

DNA unwinding occurs when a large protein complex (the pre-replication complex) binds to specific DNA sequences on chromosomes called origins of replication (ori). -The single circular chromosome of the bacterium Escherichia coli has a single ori. Unwinding of the DNA at the ori results in the formation of a replication bubble.

During elongation

During elongation, a single replication fork moves in one direction, opening up the DNA double helix. The resulting two strands will each be used as a template to synthesize a new DNA strand.

Elongation

Elongation, which involves synthesizing new strands of DNA starting from the RNA primers and using each of the parental strands as templates

primer

Following denaturation at the ori, initiation proceeds with the synthesis of a short RNA molecule called a primer. -The primer is complementary to the DNAtemplate and is synthesized one nucleotide at a time by an enzyme called primase

incorporation error rate

In eukaryotes, the incorporation error rate, which measures the probability that an incorrect base will be inserted into the new strand, is about 10−5 for DNA polymerases.

How important are spontaneous mutations as a source of mutations?

In humans, cancer is in large part a disease caused by genetic mutations. It is estimated that about 15-20 percent of cancers in humans are caused by spontaneous mutations.

mismatch

In other words, during replication, DNA polymerase will insert 1 incorrect base in 100,000, resulting in a mismatch between complementary strands (for example, placing an A across from a C)

DNA replication involves several proteins

Initiation begins when the DNA helix unwinds, or denatures, so that the replication machinery can access it.

Initiation

Initiation, which involves unwinding (denaturing) the DNA double helix to separate the two strands and synthesizing of RNA primers

antiparallel

Pertaining to molecular orientation in which a molecule or parts of a molecule have opposing directions. -These two strands are antiparallel, meaning they run in opposite directions (the free 5′ phosphate at the end of one strand sits across from the free 3′ hydroxyl of the other strand). -The two antiparallel strands twist around one another in a clockwise, or right-handed, helix, with a complete twist occurring every ten nucleotides (every 3.4 nanometers).

Proofreading

Proofreading is performed by the DNA polymerases during replication. If a DNA polymerase recognizes that it has created a mismatch, it stops, backs up, removes the mismatched nucleotide, and then recommences polymerization (FIGURE 9.15A). DNA polymerases are very good at recognizing mismatches: 99 percent are recognized and removed by the enzyme. You can think of this process as akin to spell-check on a word-processing program.

Telomeres Can Be Extended by Telomerase

Telomerase is an RNA-dependent DNA polymerase that carries an RNA template that base-pairs with the single-strand overhang left after DNA replication of the lagging strand. Extending the number of repeated sequences adds DNA sequence to the chromosome end, which can then be subsequently lost by chromosome shortening during DNA replication.

Telomeres

Telomeres are DNA sequences that do not encode proteins. Their role is to protect the important protein-coding DNA in the chromosome from being lost. -In many species, telomeres are long strings of short, repeated DNA sequences. In humans and other vertebrates, the repeated sequence is TTAGGG, and in humans it is repeated about 2,500 times.

complementary base pairs

The AT (or AU), TA (or UA), CG, and GC pairing of bases in double-stranded DNA, in transcription, and between tRNA and mRNA. -In DNA, all base pairs are either A─T or G─C, and these are termed Watson-Crick, or complementary base pairs. This base pairing explains Chargaff's rules.

After initiation, the next step of DNA synthesis is elongation.

The ability to start polymerization without a primer is a major difference between RNA polymerases (no primer required) and DNA polymerases (primer required).

A common source of mutations is replication errors

The accurate transmission of genetic information is essential for the proper functioning and even the life of a single cell or multicellular organism. However, polymerases occasionally make mistakes in assembling polynucleotide strands.

The following relationships, now known as Chargaff's rules, held for each sample:

The amount of adenine equaled the amount of thymine (A=T), and the amount of guanine equaled the amount of cytosine (G=C), but the amounts of the other nucleotides were not all equal (A≠G,A≠C,T≠G,andT≠C). -These relationships implied that the total abundance of purines (A+G) equaled the total abundance of pyrimidines (T+C).

Since shortening is impossible to avoid, why haven't linear chromosomes disappeared?

The answer is that various mechanisms to extend chromosome ends have evolved. The ends of chromosomes in eukaryotes have special structures called telomeres.

excision repair proteins

The excision repair proteins recognize damaged DNA and remove a fragment of the strand that includes the damaged nucleotide(s), and then DNA polymerase and DNA ligase fill the gap. -For some types of DNA damage, the repair pathways recognize the specific damage and repair it directly.

The double-helical structure of DNA is essential to its function

The genetic material stores heritable information—the sequence of bases in DNAcontains information that is passed from parent to offspring. The structure of DNAprovided great insight into how this occurs.

Important Info

The goal of DNA replication is to generate two identical daughter molecules from each parent DNA molecule. It is thus critical that initiation from an ori occurs only once per round of replication. -If an ori were used more than once, the DNA in that region (or the entire chromosome in the case of prokaryotes) would be replicated more than once. -Several proteins are involved in controlling the initiation of replication at ori in both prokaryotes and eukaryotes to ensure they are used only once per round of replication.

Several types of information led to the discovery of the structure of DNA

The history of how the actual structure of DNA was deciphered is worth considering, as it illustrates how different kinds of data were critical for deciphering the structure, and was a landmark in our understanding of biology.

Okazaki fragments

The lagging strand is a mess! It is synthesized in short stretches of a few hundred nucleotides (in eukaryotes) to a few thousand nucleotides (in prokaryotes) called Okazaki fragments. -Newly formed DNADNA making up the lagging strand in DNADNA replication. DNA ligase links Okazaki fragments together to give a continuous strand.

Dispersive replication

The parental molecule could end up dispersed among both strands in the two daughter molecules.

sugar-phosphate backbone

The repeating deoxyribose then phosphate groups that form a strand of a nucleic acid. -RE: Only the deoxyribose and the phosphate are involved in the covalent linkages between nucleotides, giving each strand a sugar-phosphate backbone

replication forks

The replication bubble consists of two replication forks, each of which moves away from the ori during synthesis of the new DNA strands. The replication forks are where two strands of DNA are exposed once the double helix is opened, providing access to the proteins that perform DNA replication.

Transformation was discovered by biologists working with two strains of a bacterium, termed R and S.

The researchers showed that dead S cells could transform living R cells into the S strain. However, if the DNA of dead S cells was enzymatically degraded into individual nucleotides, transformation was no longer possible. Degrading other macromolecules, including proteins, lipids, polysaccharides, and RNA, did not alter the ability of the dead S strain to transform the R strain. -Thus it was concluded that the ability to cause a heritable change in this bacterium, transforming the R strain into the S strain, was due to information carried on DNA.

DNA helicase

The separation of the two stands of DNA at each fork is catalyzed by an enzyme called DNA helicase, which uses free energy from ATP hydrolysis to break hydrogen bonds between bases on the two strands. -DNA helicase can be thought of as a wedge that is driven between the two strands of the DNA helix, forcing them apart. -The two strands are prevented from re-forming hydrogen bonds by single-strand binding proteins that bind to each strand

Conservative replication

The two parental strands could remain together (that is, could be "conserved") in one daughter molecule, while serving as a template for another daughter molecule consisting of two newly synthesized strands.

Since DNA consists of a double helix, separating the two strands results in additional twisting of the helix in front of each replication fork.

This additional twisting is relieved by an enzyme called topoisomerase.

One of these experiments, performed by Matthew Meselson and Franklin Stahl, followed DNA replication in bacteria that were grown on medium (food) containing different isotopes of nitrogen.

This experiment elegantly showed that DNA replication is semiconservative.

Spontaneous Chemical Changes Alter Base-Pairing Properties

Two types of spontaneous chemical changes that alter base-pairing properties: a tautomeric shift, which creates a structural isomer of a base (A) and deamination (the loss of an ─NH2 group) (B). In both of these examples, the altered C base can pair with T, which results in a C−G to A─T base-pair substitution if unrepaired.

telomeres have another important function

When a chromosome undergoes a double-strand break, ends are created that trigger repair pathways in the cell. There is a repair pathway that rejoins the broken ends. It is critical that this pathway does not mistakenly act on chromosome ends and join them together. (Think about what might happen during mitosis if two chromosomes were joined.) -To prevent this, special proteins bind to the telomere repeats so that they are not recognized as chromosome breaks. Telomeres thus protect chromosome ends from being treated as chromosome breaks.

The Meselson-Stahl Experiment

When centrifuged, DNA forms bands in the test tube according to density. If the semiconservative model of replication holds, first-generation daughter molecules should show intermediate density, reflecting one strand with 14N and one strand with 15Natoms. Second-generation daughter molecules should show both intermediate- and light-density molecules, reflecting half having one strand with 14N and one strand with 15N atoms, and half having both strands with only 14N atoms. The results of the experiment were consistent with semiconservative replication only.

DNA Is in Chromosomes

When dividing cells are treated with a chemical that specifically binds to DNA, staining is limited to the nucleus and, when visible, it is the chromosomes that are stained. Shown here is a group of dividing cells from onion root. Note that a few cells are in M phase, with distinct individual chromosomes visible.

Dividing Cells Double Their DNA Content during the Cell Cycle

When the DNA in dividing cells is stained and the staining intensity is used to estimate DNA content, two distinct populations of cells are seen: those in G1 (the bigger peak) and those in S, G2, or M phases (the smaller peak, with double the DNA content).

9.1 DNA is the Molecule of Inheritance

While it would take only a few years after the rediscovery of Mendel for the chromosome theory to be accepted, it would take decades to convincingly determine what molecule carries heritable information. Ultimately, both circumstantial and experimental evidence pointed to DNA as the genetic material.

base-pair substitutions

While proofreading and mismatch repair together reduce the number of mismatches that are not removed and correctly repaired, a few mismatches will lead to base-pair substitutions. A base-pair substitution is a type point mutation. -A change of a single base pair in a nucleotide sequence (eg AT to GC).

The two strands are held together by hydrogen bonds (H bonds) that form between particular pairs of bases:

adenine and thymine form two hydrogen bonds, guanine and cytosine form three hydrogen bonds.

Watson and Crick described the structure of DNA using chemical models

both then at the Cavendish Laboratory of the University of Cambridge, used model building to solve the structure of DNA -In late February of 1953, Crick and Watson built a model that established the general structure of DNA -This structure explained all the known chemical properties of DNA,and it immediately suggested explanations for its biological functions.

In addition to proofreading and mismatch repair, there is a third DNA repair mechanism:

called excision repair, which removes damaged nucleotides and replaces them with normal ones

Elucidating DNA's structure was a major research question because it would provide

insight into (1) how DNA is replicated between cell divisions, and (2) how protein sequence information is encoded.

The end replication problem is serious:

linear chromosomes get shorter every time the cell undergoes cell division.

This DNA polymerase has two catalytic activities:

removal of the RNAnucleotides one by one, and replacement with DNA nucleotides. -For every nucleotide removed from the primer on the next fragment, the DNA polymerase adds a deoxynucleotide to the 3′ end of its fragment (FIGURE 9.12B). -Eventually the RNA primer is completely removed and replaced, leaving a single unbonded break between the DNA that was at the primer location and the rest of the DNA strand synthesized from that primer.

When the amount of DNA in each cell of a population of actively dividing cells was quantified, two populations of cells were generally seen:

some cells, which were in S, G2, or M phases of the cell cycle, had twice as much DNA as most of the other cells, which were in G1

Franklin's X-ray diffraction images suggested that

the DNA molecule was long, helical, thin, of constant thickness, and consisting of two or perhaps three polymers, with nitrogenous bases on the interior.

Watson and Crick's modeling showed that

the bases are essentially perpendicular to the antiparallel strands and that adjacent bases (those next to each other on the same strand) stack like poker chips because of weak van der Waals interactions

The only differences among the four nucleotides of DNA are their nitrogenous bases:

the double-ring purines adenine (A) and guanine (G), and the single-ring pyrimidines cytosine (C) and thymine (T).

primase

An enzyme that catalyzes the synthesis of a primer for DNADNA replication.

Semiconservative replication

Each strand of the parental molecule could be used as a template for the synthesis of a new strand in each daughter molecule.

The amount of dye binding to DNA, and hence the intensity of color observed, was directly related to the amount of DNA present:

the greater the color intensity, the more DNA. The intensity of color could be measured in an instrument called a flow cytometer.

There is a relationship between telomere length and aging:

the older the cell lineage—that is, the more cell divisions it has undergone—the shorter its telomeres. Your parents' somatic cells have shorter telomeres that yours.

The requirement of a primer for DNA polymerases allowed the development of two critical molecular techniques:

the polymerase chain reaction and DNA sequencing

The termination of DNA replication occurs

when replication forks that are moving toward one another meet. -In the circular DNA of prokaryotes, this occurs at a point that is opposite the ori where several termination sequences occur. Proteins bind to these sequences to prevent replication forks from progressing into regions that are already replicated. -In eukaryotes, replication forks moving toward one another, having originated from different ori, terminate when they run into one another, or when one reaches the end of a linear chromosome, running out of template. When replication forks meet, they are prevented from progressing into already replicated regions.

Mismatches lead to base-pair substitutions in one of two ways:

when the mismatch repair complex removes the original instead of the new strand, or when the mismatch is not repaired before the next round of DNA replication -In both cases the result is that the incorrect base in the new strand gets used as a template for DNA synthesis, resulting in substitution of the original base pair with another base pair.

point mutation

—a mutation that substitutes, deletes or inserts a single base pair in a DNA molecule.

lagging strand

—is synthesized discontinuously. -The lagging template strand is oriented so that DNA polymerase adds nucleotides to the 3' end of the new strand in the direction away from the movement of the replication fork.